The ability to detect and identify trace quantities, of analytes has become increasingly important in virtually every scientific discipline, ranging from part per billion analyses of pollutants in sub-surface water to analysis of cancer treatment drugs in blood serum.
With the advancement of technologies to make and detect biomolecules, there are multiple techniques that promise biological detection with single molecule sensitivity. However, many of these techniques have not yet found commercial applications or feasibility. The main reasons are the complexity associated with these ultra-sensitive methods, the costs, and the potential biohazards associated with the reagents. Many methods require multiple steps of chemical treatments, bulky and expensive instruments, and/or extreme care in sample handling and observation. These are not ideal for practical applications that require easy and reliable measurements that are flexible enough for user's needs.
Additionally, many of the currently used methods of detecting bioanalytes rely on markers or “tags” that bind to the bioanalytes and are detected, thereby indirectly detecting the bioanalytes(s) of interest. However, the markers or tags such as radioisotope-labeled probes, or fluorescent markers, can lose their signal intensity over time. For example, radioisotopes commonly used as “tags” or “markers” decay over time, causing a gradual loss of signal that can be detected. Because of this, some experiments need to be conducted rapidly before the signal decays beyond the limits of detection. Similarly, fluorescent probes are subject to “photobleaching” wherein exposure to ambient light causes the fluorescent probe to bleach or fade away. Again, often experiments need to be conducted quickly before photobleaching occurs, or inconveniently in a dark setting so as to avoid photobleaching.
Safety is another consideration. Radioactive labels and their required reagents must be used in carefully monitored situations due to their known biologic hazards. Radioactive wastes produced from common detection methods must be carefully disposed of so as to avoid environmental contamination. Similarly, the toxicity of cadmium in quantum dots and relatively large size of dye-loaded particles have limited their applications. Although very small size (down to 10 nm in diameter) detection has been achieved for conjugated polymer particles, their signal intensity is lower than the larger fluorescent particles. Lower signal intensity makes the particles more difficult to detect with conventional techniques.
Finally, the cost of radioactive and fluorescent substances can be substantial, both in terms of acquisition, use, safety monitoring, and their proper monitoring and disposal.
Accordingly, there is a need for a reagents methods of bioanalyte detection wherein the marker to be detected exhibits little or no signal decay, and can be safely utilized in a variety of environments without posing risks to the user or to the environment. Preferably, a marker would have a high safety profile, exhibit a long (non or low-decaying) signal intensity, and be available to users at a low cost for reagent use and disposal.